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In the present study, we have characterized an atraumatic grafting technique which permits multiple, segmental, and lamina-specific injections into cervical or lumbar spinal cord. Cell injections were performed in spinally mounted rats of different ages and spinal cord size, using a micromanipulator and glass microcapillary connected to a digital microinjector. For grafting, we used human neuroteratoma (hNT) cells, BrdU-labeled rat spinal precursors or primary embryonic spinal cord neurons isolated from E14 spinal cord of the eGFP+ rat. Systematic quantification of grafted cells was performed using stereological principles of systematic random sampling and semi-automated optical Disector software. Volume reconstruction was performed using serial sections from grafted areas and custom-developed software (Ellipse) which permits "two reference points" semi-automated alignment of images, as well as volume reconstruction and calculation. By coupling these techniques, it is possible to achieve a relatively precise and atraumatic cell delivery into multiple spinal cord segments and specific spinal laminae. Consistency of the multiple grafts position in the targeted laminar areas was verified by a systematic volume reconstruction. Good survival of implanted cells for the three different cell lines used indicate that this grafting technique coupled with a systematic analysis of the individual grafting sites can represent a valuable implantation-analytical system.

Transplantation of hNT Neurons derived from the human teratocarcinoma cell-line (NTera2/D1) has been shown to ameliorate motor dysfunction in a number of injury or disease models in which the deficits are fairly localized. However, these cells have not been used before in a model with more extensive neurodegeneration. The aim of this study is to determine the effects of hNT Neuron transplants on motor neuron function in a mouse model of familial amyotrophic lateral sclerosis (FALS) in which there is a substitution of Alanine for Glycine at position 93 of the human SOD1 gene (G93A). Amyotrophic lateral sclerosis is a fatal degenerative motor neuron disease affecting the spinal cord, brainstem, and cortex. This disease clinically manifests as progressive muscular weakness and atrophy, leading to paralysis and death within 3-5 years of diagnosis. The FALS represents 10-13% of all cases. A range of behavioral tests was used to examine spontaneous locomotor activity, coordination, and muscle strength of mice. Long-term (10-11 weeks) transplantation of hNT Neurons into the L(4)-L(5) segments of the ventral horn spinal cord of FALS(G93A) mice at 7 weeks of age (before onset of overt behavioral symptoms of disease) delayed the onset of motor dysfunction for at least 3 weeks. The average lifespan of the transplanted mice was 128 days compared to 106 days for media-injected group. The last mouse in the hNT Neuron transplanted group was euthanized at 135 days of age when it display partial paralysis of the hindlimbs. Immunohistochemical analysis of the implanted spinal cords demonstrated the survival of grafted hNT Neurons and showed many healthy-appearing motor neurons near the implant site. These results suggest that hNT Neuron transplantation may be a promising therapeutic strategy for ALS.

An alternative source of cells for neural transplantation and brain repair that has many characteristics of immature neurons is the hNT neuron, derived from an embryonal human teratocarcinoma (NTera2) cell line that is terminally differentiated in vitro with retinoic acid. The majority of hNT neurons are GABAergic in cell culture. We have determined the calcium-binding protein (CBP) phenotypes of hNT neurons for three CBPs, calretinin (CR), calbindin D-28K (CB), and parvalbumin (PV), in cell culture and after transplantation into the rat striatum. In cell culture, 95% of all cell profiles were human nuclear matrix antigen (NuMA) positive. PV-positive hNT neurons constituted 50% of all neuron-like profiles, with CB+ and CR+ constituting 14 and 6% of cells, respectively. In contrast, when the striatal grafts were examined after 30 days survival using confocal microscopy, only 10% of hNT neurons immunopositive for NuMA were PV+; 19% were CB+/NuMA+, approximately the same percentage as was seen in vitro, and 82% of grafted hNT neurons were CR+. These results suggest that hNT neurons can be subdivided into at least three subpopulations based on the CBP phenotype that they express and that there is a CBP phenotypic shift following transplantation. Three related hypotheses are proposed to account for this phenotypic shift of hNT neurons after transplantation: (a) selective survival of the CR+ subpopulation of hNT neurons, (b) selective transitory quiescence of the transplanted PV+ cells due to transplantation stress, or (c) dedifferentiation of the hNT neurons following transplantation, which may allow them to respond to local environmental cues during the engraftment process., (Copyright 2001 Academic Press.)

For optimal neural transplantation using gene engineering, it might be important to control the expression of the transfected gene extrinsically as required. This strategy could be very useful for the treatment of intractable pain that responds to opioids. For this purpose, we established a genetically modified embryonal carcinoma cell line (P19) in which the expression of beta-endorphin (beta-EP) could be controlled by the addition of dexamethasone. To obtain extrinsic control, we transfected the cells with pMAMneo containing mouse MMTV-LTR as a promoter and cDNA of the artificial beta-EP. The upregulation of beta-EP, through the activation of MMTV by the administration of dexamethasone, was confirmed in vitro. Then we transplanted these cells into the subarachonoid space in rats and evaluated the analgesic potential of these cells in vivo by hot plate test and formalin test. In the rats that received beta-EP-producing cells, we observed prominent analgesic effects after the transplantation for a month. The administration of naloxone blocked these effects. Intraperitoneal injection of 100 mg/kg dexamethasone further enhanced these effects by up to two times. These data indicate obvious analgesic effects of the cells after the transplantation and the possible exogenous upregulation of transfected beta-EP gene expression in vivo. The application of this technique might provide a new therapeutic approach to various neurological diseases., (Copyright 2000 Academic Press.)